The importance of detection and analysis of ribonucleic acid (RNA) is becoming increasingly evident. For example, a large number of pathogenic mammalian viruses (e.g. SARS-CoA, Influenza virus, Measles virus, Rabies virus, Dengue fever virus, Respiratory Syncytial Virus (RSV), HIV and Hepatitis A, C-E virus) have genomes based on RNA rather than DNA. Detection and/or analysis of such RNA are potentially of great importance, yet an accepted method that is optimal for collecting, preserving/stabilizing, transporting and extracting RNA has not yet been developed.
RNA is a labile compound and the widespread adoption for routine use of RNA as an analyte in detection and analysis of RNA has been limited because of its labile nature. The sugar-phosphate backbone of RNA is particularly sensitive to breakdown (degradation, hydrolysis) by alkaline solutions. It is also sensitive to breakdown by acidic solutions. The pH of maximum stability of RNA is generally assumed to be about neutral, but this has not previously been determined precisely.
RNA can also be degraded enzymatically by endoribonucleases (e.g., pancreatic ribonuclease). Ribonuclease activity has previously been identified in human saliva (Bardon and Shugar, 1980), but the biochemical properties of this enzyme have not been well characterized. Brandon and Shugar (1980) suggest that salivary ribonuclease is pancreatic ribonuclease-like, but this has not been established.
At least in part as result of its instability RNA is often considered as an unsuitable analyte for diagnosis or detection. In the case of RNA viruses, methods have been devised for detection that do not require direct detection of RNA. For example, liquid culturing systems are used to ‘grow up’ sufficient quantities of virus/bacteria to confirm a diagnosis. Bacterial infection is typically diagnosed by direct staining and microscopic examination of samples. Electron microscopy is also used to identify bacteria and virus containing samples. In serology, diagnosis may be accomplished by detection of antibodies directed against pathogens (e.g. viruses, bacteria, parasites) in blood serum by employing indirect fluorescent antibody testing and enzyme-linked immunosorbent assays
Reverse transcriptase PCR (RT-PCR) procedures are sensitive for detecting pathogens, and in some cases before the onset of symptoms. Rapid viral diagnosis will become increasingly critical, both for the control of epidemics and for the management of patients with viral infections. Currently, an immunofluorescence assay (IFA) is considered the “gold standard” for the detection of SARS-CoA infection. However, this test requires culturing of infectious SARS virus in laboratories with biosafety level 3 (BSL-3) facilities by well-trained technician personnel. Hence, there is a need for a more convenient, economical, and low-risk method for collecting and processing infectious clinical specimens.
RNA can be extracted from most, if not all, cell types in the human body (except erythrocytes) and from a variety of cell-containing bodily fluids and/or secretions as well as tissues. In some cases, it is also be desirable to be able to obtain RNA from other sources, including feces, urine, cerebral spinal fluid, animal tissues, bone marrow aspirates, plants, plant extracts, microorganisms, virus, soil samples, sewage, wastewater, and/or foodstuffs (including milk).
Typically, once a RNA-containing sample is collected, it must either be frozen (e.g., with liquid nitrogen) or quickly transported in the unfrozen state at 4° C. to a laboratory for extraction of RNA. The requirement for rapid transportation and/or the requirement of freezing may be problematic in terms of cost and storage space. Additionally, in the case of remote locations and/or large-scale sample collection, rapid transportation and/or freezing may not be feasible. Importantly, rapid processing/testing of clinical samples may not be feasible during an epidemic; back-logged samples will likely degrade over time and/or under sub-optimal storage conditions. A simpler procedure for collecting RNA in a form that would not require the sample be frozen or transported immediately to a laboratory including equipment such as freezers, refrigerators, centrifuges, etc., would be desirable.
As noted above, there are a variety of cellular sources of RNA. Cells from the oral cavity are conveniently obtained from samples of saliva. Saliva can be collected ‘passively’ by spitting and/or ‘actively’ with the aid of implements (e.g., swabs). Nasal mucosal samples are conveniently obtained and are a rich source or epithelial and immune cells (e.g., lymphocytes). This procedure is not as invasive compared to, for example, taking of venous blood and a simple procedure based on saliva would permit self-collection by individuals with essentially no prior training. However, once collected, the time that useable RNA can be recovered may be limited because of the presence of ribonucleases in most tissues and bodily fluids.
With the increasing use of nucleic acid-based testing in human and veterinary medicine and in research, there is a need for compositions and methods that would allow RNA to be reliably recovered from bodily fluids and/or secretions and tissues. Desirably, it should be possible to be able to store the collected bodily fluid or bodily tissue at ambient temperature for prolonged periods of time, for example several days or weeks. For example, this would be advantageous where the bodily sample or bodily tissue needs to be shipped to a distant location for purification and analysis, especially in the absence of refrigeration or freezing.
Cationic compounds, such as tetradecyltrimethylammonium oxalate, have been used previously as a component in solutions used in purification of nucleic acids. US20020146677 includes tetradecyltrimethylammonium oxalate plus tartaric acid to stabilize nucleic acid in blood. However, cationic compounds, including tetradecyltrimethylammonium oxalate, have been found to be unsatisfactory in terms of ease of use and long term stability of RNA. It has been found that once the cationic detergent is bound to nucleic acids, the nucleic acids are difficult to dissolve.
In addition, it would be desirable for the amount of RNA in the collected sample to be sufficiently large to allow for the detection of low copy number RNA species such as messenger RNA and some viruses.
This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention.